We are particularly grateful to Dr. Barry Ganetzky, in who&#39;s lab the genetic screen was performed, allowing the identification and characterization of brat as a neuroprotective gene. We thank Dr. Steven Robinow for kindly providing the collection of ENU-mutagenized flies used in the genetic screen presented in this article. We thank the members of Ganetzky lab, Drs. Grace Boekhoff-Falk and David Wassarman for helpful discussions throughout the duration of this project, Ling Ling Ho and Bob Kreber for technical assistance, Dr. Aki Ikeda for the use of his microtome facility at the University of Wisconsin and Dr. Kim Lackey and the Optical Analysis Facility at the University of Alabama.

<ol>
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The authors declare no conflicts of interest.

Forward genetic screens in Drosophila have been an effective approach to identify genes involved in different biological processes, including age-dependent neuroprotection5,23,24,25. Using this strategy, we were successful in identifying brat as a novel neuroprotective gene17.
One critical step in this protocol involves the p...

Neurons are for the most part post-mitotic and incapable of dividing1,2. In most animals, neuroprotective mechanisms exist to maintain these cells throughout the organism&#39;s lifespan, especially at old age when neurons are most vulnerable to damage. Genes underlying these mechanisms can be identified in mutants exhibiting neurodegeneration, a phenotypic indicator for the loss of neuroprotection, using a forward genetic protocol. Forward genetic screens using chemical mutagens such as ethyl methanesulfonate (EMS) or N-ethyl-N-nitrosourea (ENU) are particularly useful due to the random point mutations they induce, resulting in an inherently unbiased approach that has shed light on numerous gene functions in eukaryotic model organisms3,4,5 (in contrast, X-ray mutagenesis creates DNA breaks and can result in rearrangement rather than point mutations6).
The common fruit fly Drosophila melanogaster is an ideal subject for these screens due to its high quality, well annotated genome sequence, its long history as a model organism with highly developed genetic tools, and most significantly, its shared evolutionary history with humans7,8. A limiting factor in the applicability of this protocol is early lethality caused by the mutated genes, which would prevent testing at old age9. However, for non-lethal mutations, a climbing assay, which takes advantage of negative geotaxis, is a simple, although extensive, method of quantifying impaired motor functioning10. To exhibit sufficient locomotor reactivity, flies depend on neural functions to determine direction, sense its position, and coordinate movement. The inability of flies to sufficiently climb in response to stimuli can therefore indicate neurological defects11. Once a particular defective climbing phenotype is identified, further testing using a secondary screen such as histological analysis of brain tissue, can be used to identify neurodegeneration in climbing-defective flies. Subsequent gene mapping can then be used to reveal the genomic region on the chromosome carrying the defective neuroprotective gene of interest. To narrow down the chromosomal region of interest, meiotic mapping using mutant fly lines carrying dominant marker genes with known locations on the chromosome can be performed. The marker genes serve as a reference point for the mutation as the frequency of recombination between two loci provides a measurable distance that can be used to map the approximate location of a gene. Finally, crossing the mutant lines with lines carrying balanced deficiencies on the meiotically mapped chromosomal region of interest creates a complementation test in which the gene of interest can be verified if its known phenotype is expressed5. Polymorphic nucleotide sequences in the identified gene, possibly resulting in an altered amino acid sequences, can be evaluated by sequencing the gene and comparing it to the Drosophila genome sequence. Subsequent characterization of the gene of interest can include testing of additional mutant alleles, mutation rescue experiments and examination of additional phenotypes.

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	<tbody>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:743px;">Major equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Fume hood for histology</td>
			<td style="width:195px;"></td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Light Microscope</td>
			<td style="width:195px;">Nikon</td>
			<td style="width:123px;">Eclipe E100</td>
			<td style="width:171px;">Preferred objective for imaging is X20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Imaging software</td>
			<td style="width:195px;">Nikon</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Microscope Camera</td>
			<td style="width:195px;">Nikon</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Thermal cycler</td>
			<td style="width:195px;">Eppendorf</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:743px;">Fly pushing and climbing assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">VWR&#174; Drosophila Vial, Narrow</td>
			<td style="width:195px;">VWR</td>
			<td style="width:123px;">75813-160</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">VWR&#174; General-Purpose Laboratory Labeling Tape</td>
			<td style="width:195px;">VWR</td>
			<td style="width:123px;">89097-912</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Standard mouse pad</td>
			<td style="width:195px;"></td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Stereoscope</td>
			<td style="width:195px;">Motic</td>
			<td style="width:123px;"></td>
			<td>Model SMZ-168</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">CO2 anesthesia station (Blowgun, foot valve, Ultimate Flypad)</td>
			<td style="width:195px;">Genesee Scientific</td>
			<td style="width:123px;">54-104, 59-121, 59-172</td>
			<td>Doesn&#8217;t iinclude CO2 tank</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Fine-Tip Brushes</td>
			<td style="width:195px;">SOLO HORTON BRUSHES, INC.</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Drosophila Incubator</td>
			<td style="width:195px;">VWR</td>
			<td style="width:123px;">89510-750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:743px;">Gene mapping</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">CantonS</td>
			<td style="width:195px;">Bloomington Drosophila Stock Center</td>
			<td>9517</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">w1118</td>
			<td style="width:195px;">Bloomington Drosophila Stock Center</td>
			<td>5905</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">yw&#160;</td>
			<td style="width:195px;">Bloomington Drosophila Stock Center</td>
			<td align="right">6599</td>
			<td></td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:255px;">Drosophila line used for recombination mapping</td>
			<td style="width:195px;">Bloomington Drosophila Stock Center</td>
			<td style="width:123px;">3227</td>
			<td style="width:171px;">Genotype: wg[Sp-1] J[1] L[2] Pin[1]/CyO, P{ry[+t7.2]=ftz/lacB}E3</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:255px;">CyO/sno[Sco]&#160;</td>
			<td style="width:195px;">Bloomington Drosophila Stock Center</td>
			<td style="width:123px;">2555</td>
			<td style="width:171px;">Drosophila balancer line used for recombination mapping</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Deficiency Kit for chromosome 2L</td>
			<td style="width:195px;">Bloomington Drosophila Stock Center</td>
			<td style="width:123px;">DK2L</td>
			<td>Cook et al., 2012</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:255px;">Histology analysis</td>
			<td style="width:195px;"></td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Ethanol, (100%)</td>
			<td style="width:195px;">Thermo Fischer Scientific</td>
			<td style="width:123px;">A4094</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Chloroform</td>
			<td style="width:195px;">Thermo Fischer Scientific</td>
			<td style="width:123px;">C298-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Glacial Acetic Acid</td>
			<td style="width:195px;">Thermo Fischer Scientific</td>
			<td style="width:123px;">A38-500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Fisherbrand&#8482;&#160;Premium Microcentrifuge Tubes: 1.5mL</td>
			<td style="width:195px;">Thermo Fischer Scientific</td>
			<td style="width:123px;">05-408-129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Histochoice clearing agent 1X</td>
			<td style="width:195px;">VWR Life Sciences</td>
			<td style="width:123px;">97060-934</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Harris Hematoxylin</td>
			<td style="width:195px;">VWR</td>
			<td style="width:123px;">95057-858</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Eosin</td>
			<td style="width:195px;">VWR</td>
			<td style="width:123px;">95057-848</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Thermo Scientific&#8482;&#160;Richard-Allan Scientific&#8482; Mounting Medium</td>
			<td style="width:195px;">Thermo Scientific&#8482; 4112</td>
			<td style="width:123px;">22-110-610</td>
			<td>CyO/sna[Sco]</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:255px;">Unifrost Poly-L-Lysine microscope slides, 75x25x1mm, EverMark Select Plus</td>
			<td style="width:195px;">Azer Scientific</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Fisherbrand&#8482;&#160;Cover Glasses: Rectangles</td>
			<td style="width:195px;">Fisherbrand</td>
			<td style="width:123px;">12-545M</td>
			<td>Dimensions: 24x60 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Traceable timer</td>
			<td style="width:195px;">VWR</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Slide Warmer</td>
			<td style="width:195px;">Barnstead International</td>
			<td style="width:123px;"></td>
			<td>model no. 26025</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Slide tray and racks</td>
			<td style="width:195px;">DWK Life Sciences</td>
			<td style="width:123px;"></td>
			<td>Rack to hold 20 slides</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Fisherbrand&#8482;&#160;General-Purpose Extra-Long Forceps</td>
			<td style="width:195px;">Fisherbrand</td>
			<td style="width:123px;">10-316A</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Kimwipes&#8482;</td>
			<td style="width:195px;">Kimberly-Clark&#8482; Professional&#160;</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">6 inch Puritan applicators</td>
			<td style="width:195px;">Hardwood Products Company, Guilford, Maine</td>
			<td style="width:123px;">807-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">VWR&#174; Razor Blades</td>
			<td style="width:195px;">VWR</td>
			<td style="width:123px;">55411-050</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Tupperware or glass containers for histology liquids</td>
			<td style="width:195px;"></td>
			<td style="width:123px;"></td>
			<td>16 + 1 for running water</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">High Profile Coated Microtome Blades</td>
			<td style="width:195px;">VWR</td>
			<td style="width:123px;">95057-834</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Corning&#8482;&#160;Round Ice Bucket with Lid, 4L</td>
			<td style="width:195px;">Corning&#8482;</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Beaker</td>
			<td style="width:195px;"></td>
			<td style="width:123px;"></td>
			<td>Or other container for ice water and cassettes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Tissue Bath</td>
			<td style="width:195px;">Precision Scientific Company</td>
			<td style="width:123px;">66630</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Microtome</td>
			<td style="width:195px;">Leica Biosystems</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:255px;">Molecular analysis</td>
			<td style="width:195px;"></td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Wizard&#174; SV Gel and PCR Clean-Up System</td>
			<td style="width:195px;">Promega</td>
			<td style="width:123px;">A9282</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Ex Taq DNA polymerase</td>
			<td style="width:195px;">TaKaRa</td>
			<td style="width:123px;"></td>
			<td>5 U/&#956;l</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Invitrogen&#8482;&#160;SYBR&#8482; Safe&#8482; DNA Gel Stain&#160;</td>
			<td style="width:195px;">&#160;Invitrogen&#8482;</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">UltraPure&#8482; Agarose</td>
			<td style="width:195px;">&#160;Invitrogen&#8482;</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">1 Kb Plus DNA Ladder</td>
			<td style="width:195px;">&#160;Invitrogen&#8482;</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">ApE-A plasmid Editor software</td>
			<td style="width:195px;"></td>
			<td style="width:123px;"></td>
			<td style="width:171px;">Available for free download</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:255px;">Statistical analysis</td>
			<td style="width:195px;"></td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">R software package</td>
			<td style="width:195px;"></td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:255px;">Further analysis</td>
			<td style="width:195px;"></td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">y[1] w[*]; wg[Sp-1]/CyO; Dr[1]/TM3, Sb[1]</td>
			<td style="width:195px;">Bloomington Drosophila Stock Center</td>
			<td style="width:123px;">59967</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo forward genetic screen to identify novel neuroprotective genes in drosophila melanogaster

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic screening using chemical mutagens is a useful strategy for mapping mutant phenotypes to genes among Drosophila and other model organisms that share conserved cellular pathways with humans. If the mutated gene of interest is not lethal in early developmental stages of flies, a climbing assay can be conducted to screen for phenotypic indicators of decreased brain functioning, such as low climbing rates. Subsequently, secondary histological analysis of brain tissue can be performed in order to verify the neuroprotective function of the gene by scoring neurodegeneration phenotypes. Gene mapping strategies include meiotic and deficiency mapping that rely on these same assays can be followed by DNA sequencing to identify possible nucleotide changes in the gene of interest.

In this aerticle, we present the steps used to identify the gene brain tumor (brat) as playing a role in maintenance of neuronal integrity (e.g., neuroprotection) in adult flies17; a methodology that can be used to identify genes involved in neuroprotection. We used a forward genetic approach (the strategy is outlined in Figure 1A) to screen through a collection of chemically mutagenized flies using a climbing assay (...

Watch this Scientific Journal Video about In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster at JoVE.com

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

We present a protocol using a forward genetic approach to screen for mutants exhibiting neurodegeneration in Drosophila melanogaster. It incorporates a climbing assay, histology analysis, gene mapping and DNA sequencing to ultimately identify novel genes related to the process of neuroprotection.

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic ...

in-vivo-forward-genetic-screen-to-identify-novel-neuroprotective

Research

JoVE Journal

Genetics

9.3K Views.  University of Alabama. We present a protocol using a forward genetic approach to screen for mutants exhibiting neurodegeneration in Drosophila melanogaster. It incorporates a climbing assay, histology analysis, gene mapping and DNA sequencing to ultimately identify novel genes related to the process of neuroprotection.

Video: In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the replacement of the entire gene with a selectable marker via homologous recombination. During homologous recombination, exchange of DNA takes place between sequences with high similarity. Therefore, linear genomic sequences flanking a target gene can be used to specifically direct a selectable marker to the desired integration site. Blunt ends of the deletion construct activate the cell&#39;s DNA repair systems and thereby promote integration of the construct either via homologous recombination or by non-homologous-end-joining. In organisms with efficient homologous recombination, the rate of successful gene deletion can reach more than 50% making this strategy a valuable gene disruption system. The smut fungus Ustilago maydis is a eukaryotic model microorganism showing such efficient homologous recombination. Out of its about 6,900 genes, many have been functionally characterized with the help of deletion mutants, and repeated failure of gene replacement attempts points at essential function of the gene. Subsequent characterization of the gene function by tagging with fluorescent markers or mutations of predicted domains also relies on DNA exchange via homologous recombination. Here, we present the U. maydis strain generation strategy in detail using the simplest example, the gene deletion.

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

We describe a robust gene replacement strategy to genetically manipulate the smut fungus Ustilago maydis. This protocol explains how to generate deletion mutants to investigate infection phenotypes. It can be extended to modify genes in any desired way, e.g., by adding a sequence encoding a fluorescent protein tag.

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the ...

Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts

Controlling the expression or activity of specific genes through the myocardial delivery of genetic materials in murine models permits the investigation of gene functions. Their therapeutic potential in the heart can also be determined. There are limited approaches for in vivo molecular intervention in the mouse heart. Recombinant adeno-associated virus (rAAV)-based genome engineering has been utilized as an essential tool for in vivo cardiac gene manipulation. The specific advantages of this technology include high efficiency, high specificity, low genomic integration rate, minimal immunogenicity, and minimal pathogenicity. Here, a detailed procedure to construct, package, and purify the rAAV9 vectors is described. Subcutaneous injection of rAAV9 into neonatal pups results in robust expression or efficient knockdown of the gene(s) of interest in the mouse heart, but not in the liver and other tissues. Using the cardiac-specific TnnT2 promoter, high expression of GFP gene in the heart was obtained. Additionally, target mRNA was inhibited in the heart when a rAAV9-U6-shRNA was utilized. Working knowledge of rAAV9 technology may be useful for cardiovascular investigations.

In this manuscript, a method to prepare recombinant adeno-associated virus 9 (rAAV9) vectors to manipulate gene expression in the mouse heart is described.

Controlling the expression or activity of specific genes through the myocardial delivery of genetic materials in murine models permits the investigation ...

Quantifying Abdominal Pigmentation in Drosophila melanogaster

Pigmentation is a morphologically simple but highly variable trait that often has adaptive significance. It has served extensively as a model for understanding the development and evolution of morphological phenotypes. Abdominal pigmentation in Drosophila melanogaster has been particularly useful, allowing researchers to identify the loci that underlie inter- and intraspecific variations in morphology. Hitherto, however, D. melanogaster abdominal pigmentation has been largely assayed qualitatively, through scoring, rather than quantitatively, which limits the forms of statistical analysis that can be applied to pigmentation data. This work describes a new methodology that allows for the quantification of various aspects of the abdominal pigmentation pattern of adult D. melanogaster. The protocol includes specimen mounting, image capture, data extraction, and analysis. All the software used for image capture and analysis feature macros written for open-source image analysis. The advantage of this approach is the ability to precisely measure pigmentation traits using a methodology that is highly reproducible across different imaging systems. While the technique has been used to measure variation in the tergal pigmentation patterns of adult D. melanogaster, the methodology is flexible and broadly applicable to pigmentation patterns in myriad different organisms.

Quantifying Abdominal Pigmentation in Drosophila melanogaster

This work presents a method to quickly and precisely quantify the abdominal pigmentation of Drosophila melanogaster using digital image analysis. This method streamlines the procedures between phenotype acquisition and data analysis and includes specimen mounting, image acquisition, pixel value extraction, and trait measurement.

Pigmentation is a morphologically simple but highly variable trait that often has adaptive significance. It has served extensively as a model for ...

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in humans, but more tools and research efforts are needed to better elucidate the mechanisms involved. For over a century, the genetically highly tractable model of Drosophila has been instrumental in the discovery of key genes and molecular pathways that proved to be highly conserved across species. Many biological processes and disease mechanisms are functionally conserved in the fly, such as development (e.g., body plan, heart), cancer, and neurodegenerative disease. Recently, the study of obesity and secondary pathologies, such as heart disease in model organisms, has played a highly critical role in the identification of key regulators involved in metabolic syndrome in humans.
Here, we propose to use this model organism as an efficient tool to induce obesity, i.e., excessive fat accumulation, and develop an efficient protocol to monitor fat content in the form of TAGs accumulation. In addition to the highly conserved, but less complex genome, the fly also has a short lifespan for rapid experimentation, combined with cost-effectiveness. This paper provides a detailed protocol for High Fat Diet (HFD) feeding in Drosophila to induce obesity and a high throughput triacylglyceride (TAG) assay for measuring the associated increase in fat content, with the aim to be highly reproducible and efficient for large-scale genetic or chemical screening. These protocols offer new opportunities to efficiently investigate regulatory mechanisms involved in obesity, as well as provide a standardized platform for drug discovery research for rapid testing of the effect of drug candidates on the development or prevention of obesity, diabetes and related metabolic diseases.

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

This is a high fat diet feeding protocol to induce obesity in Drosophila, a model for understanding fundamental molecular mechanisms implicated in lipotoxicity. It also provides a high throughput triacylglyceride assay for measuring fat accumulation in Drosophila and potentially other (insect) models under various dietary, environmental, genetic or physiological conditions.

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in ...

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

In research models of liver cancer, regeneration, inflammation, and fibrosis, flexible systems for in vivo gene expression and silencing are highly useful. Hydrodynamic tail vein injection of transposon-based constructs is an efficient method for genetic manipulation of hepatocytes in adult mice. In addition to constitutive transgene expression, this system can be used for more advanced applications, such as shRNA-mediated gene knock-down, implication of the CRISPR/Cas9 system to induce gene mutations, or inducible systems. Here, the combination of constitutive CreER expression together with inducible expression of a transgene or miR-shRNA of choice is presented as an example of this technique. We cover the multi-step procedure starting from the preparation of sleeping beauty-transposon constructs, to the injection and treatment of mice, and the preparation of liver tissue for analysis by immunostaining. The system presented is a reliable and efficient approach to achieve complex genetic manipulations in hepatocytes. It is specifically useful in combination with Cre/loxP-based mouse strains and can be applied to a variety of models in the research of liver disease.

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

Hydrodynamic tail vein injection of transposon-based integration vectors enables stable transfection of murine hepatocytes in vivo. Here, we present a practical protocol for transfection systems that enables the long-term constitutive expression of a single transgene or combined constitutive and doxycycline-inducible expression of a transgene or miR-shRNA in the liver.

In research models of liver cancer, regeneration, inflammation, and fibrosis, flexible systems for in vivo gene expression and silencing are highly ...

Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System (REQS)

Drosophila melanogaster is a new model organism for studies in exercise biology. To date, two main exercise systems, the Power Tower and the Treadwheel have been described. However, a method to measure the amount of additional animal activity induced through the exercise treatment has been lacking. The Rotating Exercise Quantification System (REQS) fills this need, providing a measure of animal activity for animals experiencing rotational exercise. This protocol details how to use the REQS to assess animal activity during rotational exercise and illustrates the type of data that can be generated. Here, we demonstrate how the REQS is used to measure sex- and strain-specific differences in exercise induced activity. The REQS can also be used to evaluate the impact of various other experimental parameters such as age, diet, or population size on exercise induced activity. In addition, it can be used to compare the efficacy of different exercise training protocols. Importantly, it provides an opportunity to standardize exercise treatments between strains, allowing the researcher to achieve equal amounts of activity between groups if needed. Thus, the REQS is a notable new resource for exercise biologists working with the Drosophila model system and complements existing exercise systems.

Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System REQS

The Rotating Exercise Quantification System (REQS) can induce exercise in Drosophila melanogaster through rotation while simultaneously measuring the amount of activity performed by the animals. Here, we present a point-by-point protocol detailing how to measure activity levels of animals experiencing rotational exercise treatments using the REQS.

Drosophila melanogaster is a new model organism for studies in exercise biology. To date, two main exercise systems, the Power Tower and the Treadwheel ...

The TreadWheel: Interval Training Protocol for Gently Induced Exercise in Drosophila melanogaster

The incidence of complex metabolic diseases has increased as a result of a widespread transition towards lifestyles of increased caloric intake and lowered activity levels. These multifactorial diseases arise from a combination of genetic, environmental, and behavioral factors. One such complex disease is Metabolic Syndrome (MetS), which is a cluster of metabolic disorders, including hypertension, hyperglycemia, and abdominal obesity. Exercise and dietary intervention are the primary treatments recommended by doctors to mitigate obesity and its subsequent metabolic diseases. Exercise intervention, in particular aerobic interval training, stimulates favorable changes in the common risk factors for Type 2 Diabetes Mellitus (T2DM), Cardiovascular Disease (CVD), and other conditions. With the influx of evidence describing the therapeutic effect exercise has on metabolic health, establishing a system that models exercise in a controlled setting provides a valuable tool for assessing the effects of exercise in an experimental context. Drosophila melanogaster is a great tool for investigating the physiological and molecular changes that result from exercise intervention. The flies have short lifespans and similar mechanisms of metabolizing nutrients when compared to humans. To induce exercise in Drosophila, we developed a machine called the TreadWheel, which utilizes the fly&#39;s innate, negative geotaxis tendency to gently induce climbing. This enables researchers to perform experiments on large cohorts of genetically diverse flies to better understand the genotype-by-environment interactions underlying the effects of exercise on metabolic health.

The TreadWheel: Interval Training Protocol for Gently Induced Exercise in Drosophila melanogaster

The TreadWheel uses rotational motion to gently induce exercise in adult Drosophila melanogaster by exploiting flies&#39; innate, negative geotaxis. It allows for analysis of the interactions between exercise and factors, such as genotype, sex, and diet, and their effect on physiological and molecular assays to assess metabolic health.

The incidence of complex metabolic diseases has increased as a result of a widespread transition towards lifestyles of increased caloric intake and ...

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human genetics research. Although a number of in silico algorithms have been developed to predict the pathogenicity of variants identified in these datasets, functional studies are critical to determining how specific genomic variants affect protein function, especially for missense variants. In the Undiagnosed Diseases Network (UDN) and other rare disease research consortia, model organisms (MO) including Drosophila, C. elegans, zebrafish, and mice are actively used to assess the function of putative human disease-causing variants. This protocol describes a method for the functional assessment of rare human variants used in the Model Organisms Screening Center Drosophila Core of the UDN. The workflow begins with gathering human and MO information from multiple public databases, using the MARRVEL web resource to assess whether the variant is likely to contribute to a patient&#39;s condition as well as design effective experiments based on available knowledge and resources. Next, genetic tools (e.g., T2A-GAL4 and UAS-human cDNA lines) are generated to assess the functions of variants of interest in Drosophila. Upon development of these reagents, two-pronged functional assays based on rescue and overexpression experiments can be performed to assess variant function. In the rescue branch, the endogenous fly genes are &#34;humanized&#34;&#160;by replacing the orthologous Drosophila gene with reference or variant human transgenes. In the overexpression branch, the reference and variant human proteins are exogenously driven in a variety of tissues. In both cases, any scorable phenotype (e.g., lethality, eye morphology, electrophysiology) can be used as a read-out, irrespective of the disease of interest. Differences observed between reference and variant alleles suggest a variant-specific effect, and thus likely pathogenicity. This protocol allows rapid, in vivo assessments of putative human disease-causing variants of genes with known and unknown functions.

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

The goal of this protocol is to outline the design and performance of in vivo experiments in Drosophila melanogaster to assess the functional consequences of rare gene variants associated with human diseases.

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human ...

Dissection of Drosophila melanogaster Flight Muscles for Omics Approaches

Drosophila flight muscle is a powerful model to study diverse processes such as transcriptional regulation, alternative splicing, metabolism, and mechanobiology, which all influence muscle development and myofibrillogenesis. Omics data, such as those generated by mass spectrometry or deep sequencing, can provide important mechanistic insights into these biological processes. For such approaches, it is beneficial to analyze tissue-specific samples to increase both selectivity and specificity of the omics fingerprints. Here we present a protocol for dissection of fluorescent-labeled flight muscle from live pupae to generate highly enriched muscle samples for omics applications. We first describe how to dissect flight muscles at early pupal stages (&#60;48 h after puparium formation [APF]), when the muscles are discernable by green fluorescent protein (GFP) labeling. We then describe how to dissect muscles from late pupae (&#62;48 h APF) or adults, when muscles are distinguishable under a dissecting microscope. The accompanying video protocol will make these technically demanding dissections more widely accessible to the muscle and Drosophila research communities. For RNA applications, we assay the quantity and quality of RNA that can be isolated at different time points and with different approaches. We further show that Bruno1 (Bru1) is necessary for a temporal shift in myosin heavy chain (Mhc) splicing, demonstrating that dissected muscles can be used for mRNA-Seq, mass spectrometry, and reverse transcription polymerase chain reaction (RT-PCR) applications. This dissection protocol will help promote tissue-specific omics analyses and can be generally applied to study multiple biological aspects of myogenesis.

Dissection of Drosophila melanogaster Flight Muscles for Omics Approaches

Drosophila flight muscle is a powerful model to study transcriptional regulation, alternative splicing, metabolism, and mechanobiology. We present a protocol for dissection of fluorescent-labeled flight muscle from live pupae to generate highly enriched samples ideal for proteomics and deep-sequencing. These samples can offer important mechanistic insights into diverse aspects of muscle development.

Drosophila flight muscle is a powerful model to study diverse processes such as transcriptional regulation, alternative splicing, metabolism, and ...

A Suppressor Screen for the Characterization of Genetic Links Regulating Chronological Lifespan in Saccharomyces cerevisiae

Aging is the time dependent deterioration of an organism&#8217;s normal biological processes that increases the probability of death. Many genetic factors contribute to alterations in the normal aging process. These factors intersect in complex ways, as evidenced by the wealth of documented links identified and conserved in many organisms. Most of these studies focus on loss-of-function, null mutants that allow for rapid screening of many genes simultaneously. There is much less work that focuses on characterizing the role that overexpression of a gene in this process. In the present work, we present a straightforward methodology to identify and clone genes in the budding yeast, Saccharomyces cerevisiae, for study in suppression of the short-lived chronological lifespan phenotype seen in many genetic backgrounds. This protocol is designed to be accessible to researchers from a wide variety of backgrounds and at various academic stages. The SIR2 gene, which codes for a histone deacetylase, was selected for cloning in the pRS315 vector, as there have been conflicting reports on its effect on the chronological lifespan. SIR2 also plays a role in autophagy, which results when disrupted via the deletion of several genes, including the transcription factor ATG1. As a proof of principle, we clone the SIR2 gene to perform a suppressor screen on the shortened lifespan phenotype characteristic of the autophagy deficient atg1&#916; mutant and compare it to an otherwise isogenic, wild type genetic background.

A Suppressor Screen for the Characterization of Genetic Links Regulating Chronological Lifespan in Saccharomyces cerevisiae

Here is a protocol to identify genetic interactions through an increased copy number suppressor screen in Saccharomyces cerevisiae. This method allows researchers to identify, clone, and test suppressors in short-lived yeast mutants. We test the effect of the copy number increase of SIR2 on lifespan in an autophagy null mutant.

Aging is the time dependent deterioration of an organism&#8217;s normal biological processes that increases the probability of death. Many genetic factors ...